Inflammasome responses. Inflammasome responses. (A and B) LPS-primed peritoneal macrophages were stimulated with ATP, MSU, nigericin, or R837 for 1 h; transfected with poly (dA:dT) for 8 h; or infected with S. typhimurium, E. coli, S. aureus, or L. monocytogenes for 8 h. The concentrations of IL-1β [(A), averages of 8 WT mice and 5 PYNOD-KO mice] and LDH [(B), averages of 10 WT mice and 7 KO mice] in the culture supernatants were then determined. (C) LPS-primed peritoneal macrophages were cultured with or without nigericin for 1 h. Cells were permeabilized and stained for ASC (green) and nuclei (DAPI, blue), and observed under a fluorescence microscope. Boxed areas in the middle images are enlarged at left. Scale bars, 30 μm. Arrowheads at right indicate ASC specks. The percentage of ASC speck–positive cells in nigericin-treated cells was determined by examining more than 200 (219–743) cells, and the averages obtained using seven WT and seven PYNOD-KO mice are shown in the bar graph at right. (D) LPS-primed peritoneal macrophages were stimulated with nigericin (shaded area) or unstimulated (outlined area) in the presence of a FLICA for 1 h and then stained with PE–anti-F4/80 mAb. The FLICA fluorescence of F4/80+ cells was examined by flow cytometry. Upper panels are representative flow cytometry profiles. Lower bar graph shows the average mean fluorescent intensities using two WT and two PYNOD-KO mice. Similar results were obtained using three other WT and three other PYNOD-KO mice. (E) WT (n = 10) and PYNOD KO mice (n = 13) received an i.p. injection of 30 mg/kg LPS and then were monitored for 24 h. Similar results were obtained using 11 other WT and 11 other PYNOD-KO mice. (A–E) No significant differences were observed between WT and PYNOD-KO cells or mice. ***p < 0.005. n.s., not significant. Shinsuke Nakajima et al. ImmunoHorizons 2018;2:129-141 Copyright © 2018 The Authors